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Anti-hyperglyceamic Effects of Psidium guajava LINNCrude Leaf Extracts and Fractions in Alloxan- inducedDiabetic Mice.Eze Uchenna Nwabunwanne  ( robertbertrandeze1@gmail.com )

University of NigeriaEze Anthonius Anayaochukwu 

University of NigeriaUgwu Chukwuebuka Victor 

University of NigeriaOnuoha Maxwell 

University of NigeriaUbenyi Stanley Mary 

University of Nigeria

Research Article

Keywords: Type-2 diabetes mellitus, Psidium guajava, Anti-hyperglycemia activity, Phytochemicals

Posted Date: August 16th, 2021

DOI: https://doi.org/10.21203/rs.3.rs-812096/v1

License: This work is licensed under a Creative Commons Attribution 4.0 International License.   ReadFull License

Version of Record: A version of this preprint was published at Journal of Chemistry and NutritionalBiochemistry on August 31st, 2021. See the published version at https://doi.org/10.48185/jcnb.v2i2.283.

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AbstractIn Nigeria, rural inhabitants often resort to herbal remedies and dietary control for the treatment andmanagement of various forms of diabetes mellitus. This study was conducted to provide the rationale for theuse of Psidium guajava leaves as a potent traditional antidiabetic remedy. The crude leaf extracts of n-hexane,methanol, and ethyl acetate of Psidium guajava were separately prepared by cold maceration. Then, the ethylacetate crude extract of Psidium guajava leaves was fractionated by column chromatography to yield ethylacetate bulked fractions: EF-I (1–75), EF-II (76–150), and EF- III (151–250). The n-hexane, ethyl acetate, andmethanol crude leaf extracts and ethyl acetate bulked fractions (EF-I, EF-II, and EF-III) were evaluated forantidiabetic activity in alloxan-induced diabetic mice. The blood sugar levels of treated and untreated alloxan-induced diabetic mice were assayed as indices of antidiabetic effect. The phytochemical constituents of bothcrude extracts and ethyl acetate fractions of Psidium guajava leaves and the mean lethal dose (LD50) of ethylacetate crude leaf extract of Psidium guajava were determined. The mean lethal dose (LD50) of ethyl acetatecrude leaf extract was calculated to be 1500mg/kg b.w. The results indicated that oral administration of ethylacetate, n-hexane, methanol crude extract, and ethyl acetate bulked fractions of Psidium guajava leaves at adose of 100mg/kg b.w on treated groups exhibited much signi�cant[p < 0.001, p < 0.01and p < 0.05]anti-hyperglyceamic effect by ameliorating high blood sugar levels of alloxan-induced diabetic treated mice, whileEF-II and EF-III showed non-signi�cant[p > 0.05] anti-hyperglyceamic activity for the reduction in blood sugarlevels compared with the negative and positive control groups. The anti- diabetic potency of the crude leafextracts and ethyl acetate fractions were in the order; EC > HC > MC > EF-I > EF-II > EF-III. The results ofphytochemical screening of the crude extracts and ethyl acetate bulked fractions showed the presence oftannins, �avonoids, saponins, alkaloids, terpenoids, glycosides, and steroids while reducing sugar was absent.The results from this study give credence to the use of Psidium guajava as an antidiabetic agent in themanagement of diabetes mellitus.

IntroductionDiabetes mellitus elucidates a metabolic disease characterized by abnormal hyperglycemia which alters themetabolism of lipids, carbohydrates, proteins arising from insulin de�ciency or insensitivity of the target cellsto insulin secreted in the body (Expert Committee on the Diagnosis and Treatment of Diabetes Mellitus, 2003).The prevalence of diabetes mellitus (DM) cases has been on the increase worldwide in recent years. The WorldHealth Organization report released in 2000 estimated that over 171 million (2.8%) people are living withdiabetes mellitus in the global population, and this �gure has been projected to increase to 366 million (4.4%)by 2030 (Wild et al., 2004). Most especially, cases of type 2 diabetes mellitus (T2DM) have been increased incontrast to cases of type 1 (T1DM), an autoimmune disease that often occurs due to the destruction ofinsulin- producing beta cells of the pancreas, and results from a de�ciency in insulin secretion (Kaplan, 1989).On the other hand, T2DM has become a more serious problem in developing countries because of the trend ofurbanization and consequent lifestyle changes, perhaps most importantly exempli�ed by a shift to thewestern-style diet, which is high in fat (Matsuzawa, 1997). T2DM is mostly characterized by hyperglycaemia,insulin resistance (reduced insulin sensitivity), and obesity. Obesity is associated with not only T2DM but alsohyperlipidemia and hypertension. The coexistence of these diseases is well known as metabolic syndrome, ahigh-risk factor for cardiovascular disease (Reaven, 1988).  obesity results from a disequilibrium between

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energy intake and expenditure, and obesity is known to be a strong risk factor for Type-2 diabetes associatedwith insulin resistance (Bray et al., 2002).

1.1    Origin, distribution, and morphology of Guava (Psidium guajava Linnaeus)

Psidium guajava Linnaeus, widely known as guava is a member of the Myrtaceae family and asemideciduous tree that is native to tropical and subtropical countries with about 133 genera and more than3,800 species (Haida et al., 2011). It is native and widely distributed in South and Central America. The Guavaplant is now cultivated and naturalized throughout the tropics and subtropics in Africa, South Asia, SoutheastAsia, the Caribbean, subtropical regions of North America, Hawaii, New Zealand, Australia, and Spain. It is wellknown in the islands for its edible fruit. However, the plant is cultivated today from the west coast of Africa tothe Paci�c region, including India and China, with varieties originally introduced over the past 300 years fromthe United States (Joseph and Mini, 2011). The two most common varieties of guava fruits are red (variety:Porifera) and white (variety: Porifera) (Haida et al., 2011). The Guava plant is a low evergreen tree that growsup to 6 to 25 feet tall, with wide-spreading branches and square downy twigs; it is widely grown for its fruit inthe tropics. Generally, the guava plant has spread widely throughout the tropics because it thrives in a varietyof soils, propagates easily, and bears fruit quickly. The guava berry is an important tropical fruit that is mostlyconsumed fresh. The fruit consists of a �eshy pericarp and a seed cavity with �eshy pulp and numerous smallseeds of varying hardness depending on the cultivar (Jimenez-Escrig et al., 2001, Lapik et al., 2005; Lozoya etal., 2002). The �owers are white, incurved petals, 2 or 3 in the leaf axils; they are fragrant, with four to sixpetals and yellow anthers. The fruit is small, 3 to 6 cm long, pear-shaped, reddish-yellow, or creamy- whitewhen ripe (Haida et al., 2011). It is commonly used as food and processed as juice and jam. The guava fruitsare either eaten fresh or made into drinks, ice cream, and preserves. Guava fruit is still enjoyed as a sweet treatby indigenous peoples throughout the rainforest, and the leaves and bark of the guava tree have a long historyof medicinal uses that are still employed today (Nwinyi et al., 2008). The tree is easily identi�ed by itsdistinctive thin, smooth, copper-colored bark that �akes off, showing a greenish layer beneath. It is the hardestamong tropical fruit trees and excels most of the other fruit crops in productivity and adaptability. Moreover,guava scores over other fruits in ascorbic acid, pectin, and other mineral contents. Guava cultivars, however,display a great diversity in tree size, bearing habit, and yield, as well as in fruit size, shape, quality, and ripeningseason (Joseph and Mini, 2011).

Taxonomic Classi�cation of Psidium guajava Linnaeus. Kingdom: Plantae

Sub kingdom: Viridaephytae (green plants) Infra kingdom: Streptophytae (land plants) Division: Tracheophyta(vascular plants) Subdivision: Spermatophyta (seed plants)

Infra division: Angiospermae (�owering plants) Class: Magnoliopsida (dicotyledons) Superorder: Rosidae

Order: Myrtales

Family: Myrtaceae Genius: Psidium Linnaeus Species: guajava Linnaeus

Variety: Psidium guajava pyrifera (white guava)

1.2    Pharmacological Properties of Psidium guajava Linn.

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Different parts of the guava tree have been shown to possess various pharmacological activities due to theiruse in folk medicine for the treatment of different ailments. The potential pharmacologic activities of theextract from the fruit, leaf, bark, or roots include antioxidant, hepatoprotective, anti-allergy, anti-microbial, anti-genotoxic, anti-plasmodial, cytotoxic, antispasmodic, cardioactive, anti-cough, anti-diabetic, anti- in�ammatory,and anti-conceptive activities in vitro and animal models (Gutiérrez et al., 2008). The extract of the whole plantof Psidium Guajava excluding roots was reported to possess antibacterial, anti-fungal, anti- viral,hypoglycaemic, and diuretic activities (Tripathi et al., 1981). In other studies, anti-diarrhoeal (Lutterodt, 1989),anti-pyretic (Olajide et al., 1999; Shinha et al., 2000), antihypertensive (Ayub et al., 2010) and bio-antimutagenic (Matsuo et al., 1994) properties of guava leaf extract have been demonstrated. Interestingly,guava leaves have also attracted attention as a folk remedy for diabetes not only in Japan and East Asia(Banu et al., 2012; Nien-yung and Kuanghsiung, 2004) but also in Africa (Ojewole, 2005). Based on the need tomaintain and promote good health and prevent lifestyle-associated diseases, the Japanese Ministry of Health,Labor and Welfare published "Foods for Speci�ed Health Uses" (FOSHU), which are foods whose

 Claims of physiological effects on the human body have been o�cially approved and such foods were legallypermitted to be used as dietary products for health preservation (Arai et al., 2008). Guava leaf tea,Bansoureicha that contains aqueous guava leaf extract was approved as FOSHU and recommended forindividuals with pre- diabetes, the product has become widely accepted and commercially available in Japan(Ishida, 2001).

1.2.1    Antioxidant properties of Guava

Antioxidants are substances that can prevent or reduce the oxidative damage of biomolecules (lipids, proteins,and nucleic acids) by reactive oxygen free radicals such as superoxide, hydroxyl, peroxyl, alkoxyl, andnonradicals such as hydrogen peroxide, hypochlorous, etc.  guava, compared with other fruits and vegetables,is also rich in antioxidants that help to reduce the incidence of degenerative diseases such as arthritis,arteriosclerosis, cancer, brain dysfunction, heart disease, and in�ammation (Feskanich et al., 2000). Besidespreventing or delaying oxidative damage of these essential biomolecules like lipids, proteins, and nucleic acidscaused by reactive oxygen species, antioxidants were reported to retard aging (Feskanich et al., 2000; Gordon,1996; Halliwell, 1996). They scavenge radicals by inhibiting the initiation and breaking of chain reactions,suppressing the formation of free radicals by binding to metal ions, reducing hydrogen peroxide, andquenching superoxide and singlet oxygen (Shi et al., 2001). Studies have shown that the most abundantantioxidants present in fruits are polyphenols and ascorbic acid, and polyphenols, which contain a signi�cantamount of �avonoids which are mainly present in ester and glycoside forms (Fleuriet and Macheix, 2003). Inthe case of guava, free ellagic acid and glycosides of myricetin and apigenin are found to be present (Koo andMohamed, 2001). Studies have shown that guava fruit and leaves possess antioxidant and free radicalscavenging capacity (Chen et al., 2007).

1.2.2    Anti-hyperlipidemic activity of Guava

It has been reported that streptozotocin-induced diabetic rats treated with raw peel extract of P. guajava fruitfor twenty-one days showed a signi�cant decrease in serum triglycerides, total cholesterol, very-low-densitylipoprotein (VLDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and an increase in serum high-

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density lipoprotein (HDL) cholesterol (Rai et al.,2010). A long-term clinical trial that investigated the effects ofthe consecutive intake of guava for eight weeks on the parameters of hyperlipidemia, diabetes, and safety intwenty-three subjects with borderline or mild hyperlipidemia with or without type 2 diabetes mellitus showedmuch signi�cant reduction in total serum cholesterol level, serum triglycerides level in subjects with hyper-triglyceamia and that of phospholipid in subjects with hyper-phospholipidemia. In addition, the extractsigni�cantly reduced blood glycosylated haemoglobin(HbA1c%) and raised serum adiponectin levels in eachsubject with adiponectinemia and hyperglycemia conditions while levels of high-density lipoproteincholesterol, non-esteri�ed fatty acids (NEFAs), and lipid peroxide were not signi�cantly reduced (Asano et al.,2007). In addition, research studies have shown that aqueous extract of Psidium guajava containscomponents of LDL-cholesterol that exhibit anti-glycation activity, suggesting its contribution to the preventionof neurodegenerative and cardiovascular diseases (Chen et al., 2010; Hsieh et al., 2005). Studies on humanshave found that the consumption of guava for 12 weeks reduced blood pressure by 8%, total cholesterol levelsby 9%, triacylglycerides by almost 8%, and induced an 8% increase in the levels of HDL-cholesterol. Theauthors attributed these effects to the fruit's high potassium and soluble �ber contents (Singh et al., 1993;Singh et al., 1992). Daily oral administration of red (pink) guava puree supplements showed a signi�canteffect by reducing total cholesterol, low-density lipoprotein cholesterol and triglycerides levels, and resulted inan increase in high-density lipoprotein cholesterol level in high fat diet induced obese rats (Norazmir and Ayub,2010). Red (pink) guava is reported to have high crude �ber and mineral content, especially potassium,sodium, magnesium, phosphorus, zinc, and iron (Ayub et al., 2010).

 1.2.3    Hepatoprotective and Cardioprotective effects of Guava

It has been reported that aqueous extract of lyophilized guava fruit peel exhibited a heptaprotective effect indiabetic test subject by maintaining a signi�cant reduction in serum alanine aminotransferase (ALT),aspartate aminotransferase (AST), alkaline phosphatase (ALP), and creatinine levels in streptozotocin-induceddiabetic rats(Rai et al.,2010). In another study, it was noted that the oral administration of guava pulp and ratfood supplemented with guava seeds signi�cantly diminished the levels of AST and ALT enzymes in normalWistar rats (Farinazzi-Machado et al., 2012). A study of aqueous extract of P. guajava showed ahepatoprotective activity in acute experimental liver injury induced by a combined mixture of carbontetrachloride, paracetamol, and thioacetamide. The effects observed were compared with a knownhepatoprotective agent, silymarin, and histological examination of the liver tissues that supportedhepatoprotective activity (Roy et al., 2006). Other similar studies have also showed a cardioprotective effect ofaqueous extract of P. guajava in myocardial ischemia-reperfusion injury in isolated rat hearts, which wasprimarily linked to its radical-scavenging action (Yamashiro et al., 2003).

1.2.4    Anti-obese property of Guava

A study has established that obesity results from an imbalance between energy intake and expenditure, andlinked obesity as a predisposing risk factor to the development of Type-2 diabetes mellitus, which is oftenassociated with insulin resistance (Bray et al., 2002). Studies have shown that there is an increasingprevalence of obesity among persons, which poses a global health challenge and hence, linked to somediseases. Obesity is the most common nutritional disorder affecting people in the developed world and it isconsidered a risk factor associated with the development of major human diseases, including cardiovascular

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disease, diabetes, and cancer. Signi�cantly, daily consumption of fat-enriched diets often tends to promoteobesity. In addition, increased intake of high caloric (energy and fat) food promotes body fat storage, greaterbody weight, and adiposity in humans (Bray et al., 2002); and animals (Estadella et al., 2004). Over-the-counterremedies like nutritional supplements are extremely popular, especially for weight reduction, obesity, andhealthy vitality. Inhibition of digestion and absorption of dietary fats have been used as targets in obesitytreatment (Weigle, 2003). Red guava purée intake has the bene�cial effect of lowering body weight (Nzi et al.,2007; Norazmir and Ayub, 2010) and suppressing obesity in diet-induced obese rats (Norazmir and Ayub,2010). Ram et al. (1992) reported that moderate feeding of pink guava purée caused changes in dietary fattyacids and carbohydrates.

1.2.5    Anti-hypertensive activity of Guava

A study on patients with essential hypertension showed that the guava extracts administered to subjectsbefore meals in a randomized and single-blind fashion for twelve weeks led to a signi�cant net decrease inblood pressure values and a signi�cant increase in high-density lipoprotein cholesterol (HDL-C) after twelveweeks of Guava fruit substitution in hypertensive test subjects (Ram et al., 1992). Similar study has alsodemonstrated that red guava exhibits an antihypertensive property which is capable of reducing bloodpressure (Ayub et al., 2010). In addition, it was reported that polyphenols prevented cardiac hypertrophy andthe production of reactive oxygen species, as well as improved vascular function in antihypertensiveexperimental rat (Al-Awwadi et al., 2004). Red guava extract showed a signi�cant lowering effect on systolicblood pressure of high-fat diet- induced obese rats (Norazmir and Ayub, 2010).

1.2.6    Anti-diarrheal properties of guava

It is evident that guava leaf extracts and fruit juice exhibited signi�cant recovery rate among test subjectsinfected with infantile diarrhea in a clinical control experimental study. The study further reported that 62infants treated with a separate oral administration of both leaf extracts and fruit juice had a signi�cantrecoverY rate of 87.1% within 3 days. It was concluded in the study that guava extract and fruit juice have agood anti- diarrheal effect against infantile rotaviral enteritis (Wei et al., 2000).

1.2.7    Anti-mutagenic and anticancer properties of guava

Few research studies have established anti-mutagenic and anticancer activities of P. guajava leaves. Notably,it has been reported that Psidium guajava pulp, peel, and seed extracts exert anticancer effects on bothhematological and solid neoplasms, which were implicated as a result of antioxidant properties exhibited bythe plant due to the presence of antioxidant compounds (Bomtempo et al., 2012).

1.3    Aim of Study

This study investigated the anti-hyperglycemia activities of n-hexane, methanol, ethyl acetate crude leafextracts, and ethyl acetate bulked fractions of Psidium guajava in alloxan-induced diabetic mice.

1.3.1    Objectives of the Study

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(i)    To evaluate the phytochemical constituents of n-hexane, methanol, ethyl acetate crude leaf extracts, andethyl acetate bulked fractions of Psidium guajava Linn.

(ii)    To determine the mean lethal (acute) dose (LD50) of ethyl acetate crude leaf extract of Psidium guajavaLinn.

(iii)    To assess antidiabetic activities of ethyl acetate, bulk fractions (EF-I, EF-II, and EF-III), n-hexane, ethylacetate, and methanol crude leaf extracts of P. guajava to an approved reference anti-diabetic drug andnegative control.

Materials And Methods2.1    Materials

The test materials used in this research study include Wistar albino mice (animal), guava plant material,chemical reagents, and laboratory equipment.

2.2    Animal

Thirty-six (36) Wistar albino mice of body weight between 50-65g used for the study were purchased from theAnimal House of the Department of Zoology, University of Nigeria, Nsukka Campus. The mice were fed withpoultry starter feed, drinking water ad libitum, and acclimatized for seven (7) days. The mice were furtherdivided into nine (9) groups consisting of four (4) mice per group.

2.3    Drug/Chemical Reagents

All chemicals and antidiabetic drugs (Glucophage) used in this study were based on the recommendedstandard and analytical grade.

2.4    Instruments/Equipment

The equipment used for this study was domiciled at the Chemistry Department, Federal University ofAgriculture, Markurdi and the Medical Biochemistry Department, University of Nigeria, Enugu State, Nigeria.

2.5    Plant Material

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The plant leaves of Psidium guajava (Guava) used for this research study were obtained from the Botanicalgarden of the University of Nigeria, Nsukka Campus, Enugu State, Nigeria. The whole plant was identi�ed byDr. N. O Nweze, a renowned taxonomist in the Department of Plant Science and Biotechnology, University ofNigeria, Nsukka Campus, Enugu State, Nigeria.

              

2.6    Experimental design

  Thirty-six (36) Wistar albino mice (50-65g) body weight were randomly divided into nine groups comprisingfour (4) mice per group (n=4/group). Group 1 was not induced with diabetes but received diluted dimethylsulphoxide (5ml/kg). Alloxan monohydrate (Merck, Germany) was dissolved in cold normal saline andadministered intraperitoneally at a dose of 120mg/kg per body weight to groups 2, 3, 4, 5, 6, 7, 8, and 9. Thealloxanized groups were hyperglycemic when blood glucose levels rose to ≥ 120mg/dL. Group 3 received astandard antidiabetic drug (Glucophage, 5mg/kg per body weight) while group 2 received dimethyl sulphoxide(5ml/kg per body weight). The Group 4 mice received ethyl acetate bulked fraction I, EF-I (1-75) at a dose of100mg/kg per body weight. The same oral treatment was given to groups 5, 6, 7, 8, and 9 with ethyl acetatefractions; EF-II, EF-III, ethyl acetate crude extract, methanol extract, and hexane extract respectively at the samedose of 100mg/kg as showed in table 3.3.8. Oral administration of Glucophage, dimethyl sulphoxide, fractionsand crude extracts of Psidium guajava leaves lasted for 14 days, during which the blood glucoseconcentrations of the mice were measured from the 1st - 14th day. The Psidium guajava leaf extracts weredissolved in fresh dimethyl sulphoxide and administered orally. The hypoglycemic effects of the crude extractsand ethyl acetate fractions were investigated on alloxan-induced diabetic mice.

Table 2.6: Administration of Crude Extracts and Ethyl acetate bulked fractions (EF-I, EF-II, and EF-III) of PsidiumGuajava leaves in alloxan-induced diabetic mice.

Group (4 mice per group) Sample (Dosage)

Group 1 (Normal control) Dimethyl sulphoxide(DMSO) + distilled water (5ml/kg b.w)

Group 2(negative control) (DMSO) + distilled water (5ml/kg b.w)

Group 3(diabetic treated/ positive control) Glucophage (5mg/kg b.w)

Group 4(diabetic treated mice) Ethyl Acetate Fraction I(EF-I)(100mg/kg b.w)

Group 5(diabetic treated mice) Ethyl Acetate Fraction II(EF-II) (100mg/kg b.w)

Group 6( diabetic treated mice) Ethyl Acetate Fraction III(EF-III) (100mg/kg b.w)

Group 7(diabetic treated mice) Ethyl Acetate Crude Extract (100mg/kg b.w)

Group 8(diabetic treated mice) Methanol Crude Extract (100mg/kg b.w)

Group 9(diabetic treated mice) N-hexane Crude Extract (100mg/kg b.w)

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2.7        Ethical Clearance

The ethical clearance and permission to undertake this research was granted by the Research EthicsCommittee, College of Medicine, University of Nigeria, Enugu State, Nigeria. The experiment was conducted, inaccordance with the regulations of the Institutional Animal Research Ethics Committee, University of Nigeria,Enugu State, Nigeria.

2.8    Preparation of Plant Material (P. guajava)

The guava leaves were chopped and blended with a manual grinder into a �ne powdery weighing three (3kg)kilograms.

2.9    Extraction of Plant Material ((P. guajava)

The leaf extraction was carried out by cold maceration (Akah et al., 2002; Akah and Okafor, 1992). Each onekilogram (1kg) �ne powder of dried Psidium guajava leaves was soaked in a separately labeled glass �ask(500ml) containing 400ml of analytically graded n-hexane, ethyl acetate, and methanol reagent respectively.

Each preparation was �ltered through a grade 1 Whatman �lter paper, and each �ltrate was separatelycollected into a separate crucible before drying by evaporation under a steady air current for about 24 hoursuntil a soft mass (extract) was obtained. Each crude extract obtained was weighed and recorded. The extractswere carefully air-dried to remove all traces of solvents under a thermal water bath. The percentage (%) yieldsof n-hexane, ethyl acetate, and methanol crude extracts were calculated, respectively.

2.10    Column Chromatographic Fractionation of Ethyl Acetate Crude Leaf Extract (Psidium guajava) Columnglassware made up of a simple straight glass tube 60cm long with a diameter size of 3.5 cm was used forcolumn fractionation of ethyl acetate crude extract of Psidium guajava leaves. Firstly, the column glasswarewas rinsed with distilled water and later air-dried. A dried cotton wool plug was inserted into the bottom of theglass column just below the tap. The column chromatography was performed on silica gel (Machery Nagel,Germany) with a mesh pore size 70-230nm. The silica gel was packed into the glass column by the wetloading method before fractionating the ethyl acetate crude extract of Psidium guajava leaves to obtainfractionated bioactive constituents. The procedure for the gravity elution column chromatography was carriedout by Harbone (1998) protocol as described below;

I.    Column Development

The dry silica gel powder (Machery Nagel, Germany) with a mesh pore size of 70-230nm weighing 150 g wascarefully packed into the glass column by the slurry packing(wet loading) method. The slurry silica gel wasprepared by mixing dry silica gel powder and a solvent volume of 200ml n-hexane (JHD®) in a glass beaker.The mixture was properly stirred for 10 minutes until the silica gel was properly preabsorbed. A ball of cotton

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wool was plugged just above the tap and 1.5mm thick acid-treated sand (BDH®) was added and allowed tosettle onto the plugged cotton wool. The glass column was equilibrated with a few drops of n-hexane toachieve a marked level. The slurry was carefully poured into the column glassware and gently allowed to settlein the glass column. A vibrator was used to ensure that the column glass set-up was properly packed. The n-hexane solvent level was above the silica gel-packed column.

II.    Preparation of Plant Material for Column Fractionation

The ethyl acetate crude leaf extract of Psidium guajava was placed into a clean beaker and properly stirredfollowed by the addition of a solvent mixture of dimethyl ether and ethyl acetate (2:1) to preabsorb themixture, and air-dried. A �ne powder of ethyl acetate crude extract of P. guajava leaves was obtained after airdrying. The �ne powder of ethyl acetate crude extract of Psidium guajava leaves weighing 1.5g was addedonto a 1.5mm thick acid-treated sand layer (BDH, England) just above the wet silica gel in the column.

III.    Solvent System

The column fractionation was carried out using different solvent systems (mixtures) using pure analyticalgraded reagents in sequence and polarity strength.The volume ratios of n-hexane: ethyl acetate were used inthe fractionation process with varying percentage volume ratios (100%:0% –50%:50%). At the beginning ofcolumn fractionation, a solvent volume ratio of 500ml volume of n-hexane (100%): ethyl acetate (0%) wasused to elute nonpolar compounds. The sequence and polarity to volume ratios of the solvents wereconsidered to avoid column cracking and achieve e�cient separation. At the end stage of the fractionation,the column was thoroughly washed with 500ml of 99.9% methanol to obtain the methanol fraction separately.The solvent system (mixture) was continuously added and a gradual �ow rate of 10 drops per minute wasobtained.

IV.    Fraction Collection

Serial labeled glass vials (5ml) were used for collecting solvent fractions (eluents). The collection of fractionswas achieved after a successive elution to obtain a total number of two hundred and �fty (250) vial fractions.The solvent vial fractions were kept uncovered to air-dry for 24 hours after collection. The solute deposited ineach vial was redissolved with a 5ml ethyl acetate solution (JHD®, China). The vial fractions were groupedinto three main bulk fractions; EF-I, EF-II, and EF-III according to the retention factor (Rf) values of the thin layerchromatography �ngerprints (TLC) plates (Chromatogram).

2.11    Thin Layer Chromatography (TLC)

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Thin-layer chromatography (TLC) is one of the laboratory techniques applied in organic research laboratories.It is used to achieve a quick separation of organic compounds and to ascertain the level of purity of a givenmixture. TLC also helps to detect and analyze organic one or more compounds by comparing with knownsamples in terms of its purity with identi�ed samples as well as monitoring the progress of separation duringthe extraction or puri�cation process. The TLC comprises of a small glass or plastic plate coated with a thinlayer of a dry solid silica gel or alumina gel, which serves as the most common stationary phase. Mobilephase could be an organic solvent or solvent mixture. The sample mixture is spotted near the bottom of theplate as a small, and then placed in a developing chamber containing a little volume of solvent (mobilephase). The solvent travels up the plate and carries the sample mixture along with it through the capillaryaction. As a result of the solubility of various components of the mixture in the mobile phase, the strength oftheir adsorption on the stationary phase and each component in the mixture moves at a different rate. As thesolvent moves near the top of the plate (solvent front). It is allowed to evaporate, leaving behind thecomponents of the mixture at various distances from the point of origin (Dhont, 1980). The ratio of thedistance traveled by a compound to the distance traveled by the solvent is the Rf value (retention factor). Thisvalue is characteristic of the compound, the solvent, and the stationary phase (Dhont, 1980). The retentionfactor, Rf = distance traveled by sample/ distance traveled by solvent.

Procedure:

Thin-layer chromatography (TLC) was performed on a silica gel precoated plate (GF254, Merck, UK). A solventvolume ratio of n-hexane to ethyl acetate (5ml:5ml) was used as the mobile phase for each fraction; EF- I, EF-II,and EF-III. The plates were sprayed with a mixture of 20% sulphuric acid dissolved in methanol and thenheated to about 120°C for 3 minutes to visualize spots. This is suitable for the detection of most polarcompounds such as carbohydrates, etc. A total of two hundred and �fty (250) eluted fractions were furtherbulked into three main groups of ethyl acetate fractions based on the separation pattern of the constituentcompounds (Rf values) and labeled EF-I (1-75), EF-II(76-150) and EF-III(151-250) accordingly.

Results3.1    Actual and Percentage Yield of Plant Extracts and Column Fractions

The plant extraction yielded 1.5g (21% w/w) n-hexane crude extract, 3.0g (42% w/w) ethyl acetate crudeextract, and 2.51g (35% w/w) methanol crude extract of Psidium guajava Linn leaves.

Table 3.1(a): Yield of Psidium guajava Crude Leaf Extracts

S/No Type of Extract Amount of Extract (gm) % Yield

1. N-hexane 1.5 21

2. Ethyl acetate 3.0 42

3. Methanol 2.51 35

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Table 3.1(b): Bulking of Column Fractions of P. guajava Ethyl Acetate Crude LeafExtracts

 

S/No Ethyl acetate Bulked Fractions Range of Combined Fractions

1. Bulk Fraction I (EF-I) 1-75

2. Bulk Fraction II (EF-II) 76-150

3. Bulk Fraction III (EF-III) 151-250

      

 

 

 

 

   The column fractionation yielded 250 column fractions which were bulked together into three main bulkedfractions (EF- I,     EF-II, and EF-III), respectively.

 

      

      Table 3.1 (c): Retention Factor Values (Rf) of Ethyl Acetate Fractions of Psidiumguajava Leaves.

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PYTOCHEMICALCONSISTUENTS

 

ABUNDANCE

ETHYL ACETATE

BULK FRACTION(EF-I)

ETHYL ACETATE

BULK FRACTION(EF-II)

ETHYL ACETATE BULKFRACTION (EF-III)

Reducing Sugar - - -

Tannins + + +

Steroids ++ + +

Flavonoids ++ + +

Saponins ++ + -

Alkaloids ++ + +

Terpenoids ++ + +

Glycosides - - -

S/N Bulk Fractions Retention Factor(Rf ) Values

1  Bulk Fraction I(1-75) 0.33

2  Bulk Fraction II(76-150) 0.19

3 Bulk Fraction III(151-250) 0.23

 

 

 

 

 

 

 

   Table 3.1(d): Qualitative Phytochemical Screening of Ethyl Acetate Fractions of Psidium guajava Leaves.

 

 

                         *Interpretation: -Absence, + Trace, ++Moderate, +++ Abundant

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Table 3.1(e): Qualitative Phytochemical Screening of Ethyl Acetate, Methanol, and n-Hexane Crude Extracts of

Psidium guajava Leaves.

 

 

PYTOCHEMICALCONSISTUENTS

               

                                                        ABUNDANCE

 

N-HEXANE CRUDEEXTRACT  (HC)

 

ETHYLACETATE CRUDE EXTRACT (EC)

 

METHANOL CRUDE         EXTRACT  (MC)

Reducing Sugar  - - -

Tannins ++ ++ +++

Steroids ++ +++ ++

Flavonoids + +++ ++

Saponins + + +

Alkaloids + ++ +

Terpenoids + + ++

Glycosides + - -

 

3.2    Determination of Mean Acute (Lethal) Toxic Dose (LD50)

The mean acute toxic dose was determined in two phases I and II at different dosages of ethyl acetate crudeleaf extract of Psidium guajava. The acute toxicity study was determined in mice using the method of Lorke(1983). The tests involved two phases. The �rst phase was used to determine the toxic range. The threegroups 1, 2, and 3 comprising of three (3) mice was separately administered 10mg/kg b.w, 100mg/kg b.w, and1000 mg/kg b.w doses of ethyl acetate crude leaf extract solubilized in 5% (v/v) dimethyl sulphoxide. Thetreated mice were observed after 24hrs for any lethal signs, abnormal behaviour, and death. There was nolethal signs and death recorded. In the second phase of the toxicity study, each group was separatelyadministered with different doses of 1500, 2000, and 5000 mg/kg per body weight of ethyl acetate crude leafextract, respectively. The treated mice were observed after 24hrs for lethality or signs of acute intoxication. Theacute toxic dose (LD50) was calculated. The result obtained is shown below:

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     Table 3.2: Outcome of Acute Toxicity Study

 

Phase  Groups of Mice

(3 mice per group) 

Dosage (mg/kg b.w.)  Mortality  Behavioural          Changes 

Phase I  Group 1  10  0/3  Nil 

Group 2  100  0/3  Nil 

Group 3  1000  0/3  Nil 

Phase II  Group 1  1500  1/3  yes 

Group 2  2000  2/3  yes 

Group 3  5000  2/3  yes 

3.3    Anti-hyperglyceamic Effect of Column Chromatographic Fractions of Psidium guajava Ethyl AcetateCrude Extract in Alloxan-induced Diabetic Mice

3.4    Anti-hyperglyceamic Effects of Ethyl Acetate, n-Hexane, and Methanol Crude Leaf Extracts of Psidiumguajava in Alloxan-induced diabetic mice

3.5       PERCENTAGE BLOOD GLUCOSE REDUCTION ON TREATMENT DAYS

Figure 3.5.1(A and B) showed the results of percentage mean blood glucose(Mean±SEM) reduction oftreatment groups on the �rst(1st) and �fth(5th) day of oral treatment. The positive control(group 3) mice weretreated with Glucophage at a dose of 5mg/kg b.w, while groups 4, 5, and 6 were treated with ethyl acetatefractions (EF I), (EF II) and (EF III) at a dose of 100mg/kg b.w and group 7, 8 and 9 treated with ethyl acetate,methanol and n-hexane crude extracts of P. guajava leaves at a dose of 100mg/kg/b.w respectively. The oraltreatment of alloxan-induced diabetic mice with crude and partially puri�ed chromatographic fractions on Day1, group 8 mice treated with methanol crude(MC, 100mg/kg b.w) leaf extract of P. guajava showed the highestpercentage blood glucose reduction with a mean (Mean±SEM) (45%), followed by n-hexane, HC(42%) andethyl acetate crude extract (40%), EF I (39%) while EF II(18%) and EF III(10%) had least percentage bloodglucose reduction. A statistical comparison of the anti-hyperglycemia effect between all treatment groups andpositive controls using a One-way ANOVA (Dunnett's multiple) showed that each bar(Mean±SEM) was notsigni�cantly different from the positive control group(18% mean percentage blood glucose reduction). Thisimplies that the groups treated with EF I, EF II, EF III, ethyl acetate, methanol, and n-hexane crude extract of P.guajava leaves had a signi�cant anti-hyperglyceamic effect with a percentage reduction in high blood glucoselevel compared with the standard reference drug (Glucophage, 5mg/kg b.w) in the order of MC>HC>EC>EF-I>EF-II while the EF-III had a least anti-hyperglyceamic effect with percentage glucose reduction. On thesecond(2nd) day of oral treatment of alloxan-induced diabetic mice with crude and partially puri�ed

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chromatographic column fractions, group 7 mice treated with ethyl acetate crude100mg/kg b.w) leaf extractof

P. guajava showed the highest percentage blood glucose reduction with a mean (Mean ±SEM) (43%), followedby methanol, MC (40%), and n-hexane extract(39%), EF I (31%) while EF II(15%) and EF III(13%) had leastpercentage blood glucose reduction. A statistical comparison between all treatment groups and positivecontrols(group 3) using Dunnett's multiple One way ANOVA showed that each bar (Mean±SEM) was notsigni�cantly different from the positive control group(40% mean percentage blood glucose reduction). Thisimplies that the groups treated with EA fraction I, EA fraction II, EA fraction III, ethyl acetate, methanol, and n-hexane crude extract of P. guajava leaves had a signi�cant anti-hyperglyceamic effect compared with thestandard reference drug (Glucophage) administered at a dose of 5mg/kg b.w in the order of EC>MC>HC>EF- I>EF-II while the EF-III had least percentage blood glucose reduction.

Figure 3.5.2 (A and B) showed the results of mean percentage blood glucose reduction (mean± SEM)for alltreatment groups on the eighth (8th) and eleventh (11th) day of oral treatment with reference drug(Glucophage,5mg/kg b.w), crude extracts(n-hexane, ethyl acetate, methanol) and ethyl acetate fractions (EF-I,EF-II and EF-III) of P. guajava leaves respectively. On the eighth (8th) day of oral treatment of alloxan- induceddiabetic mice with crude and partially puri�ed chromatographic fractions. Group 7 mice treated with ethylacetate(EC, 100mg/kg b.w) crude leaf extract of P. guajava showed the highest percentage mean bloodglucose reduction with a mean(Mean±SEM) (43%), followed by methanol, MC(39%) and n-hexane extract, HC(30%), EF I (25%), EF III(5%) while EF II showed least mean percentage blood glucose reduction. A statisticalcomparison of the anti-hyperglycemia effect of all treatment groups and positive controls using One-wayANOVA (Dunnett's multiple) showed that each bar (Mean±SEM) was not signi�cantly different from thepositive control group(40%mean percentage blood glucose reduction). This implies that the groups treatedwith EA fraction I, EA fraction II, ethyl acetate, methanol, and n-hexane crude leaf extract of P.guajava had asigni�cant anti-hyperglycemia effect compared with the standard reference drug(Glucophage administered ata dose of 5mg/kg b.w) in the order of EC>MC>HC>EF-I >EF III>EF-II, even though EA fraction II(EF-II) had leastpercentage blood glucose reduction. On the eleventh (11th) day of oral treatment of alloxan-induced diabeticmice with crude and partially puri�ed chromatographic fractions. The group 7 mice treated with ethyl acetatecrude (100mg/kg b.w) leaf extract of P. guajava showed the highest percentage blood glucose reduction witha mean (Mean±SEM) (40%), followed by methanol(38%) and n-hexane extract(30%), EF I (25%) while EFII(18%) had least percentage blood glucose reduction and EF III had -5% with no signi�cant mean bloodglucose reduction. A statistical comparison of the anti-hyperglycemia effect between all treatment groups andpositive controls using One-way ANOVA (Dunnett's multiple analysis) showed that each bar (Mean±SEM) wasnot signi�cantly different from the positive control group(42% mean percentage blood glucose reduction). Thisimplies that all treatment groups treated with EF I, EF II, EF III, ethyl acetate, methanol and n-hexane crudeextract of P.guajava leaves at a dose of 100mg/kg b.w had a signi�cant anti-hyperglycemia effect comparedwith the standard reference drug (Glucophage) administered at a dose of 5mg/kg b.w in the order ofEC>MC>HC>EF-I >EF-II >EF-III.

Figure 3.5.3 showed the result of percentage blood glucose reduction (Mean±SEM) for the treatment groupson the fourteenth (14th) day of oral treatment of alloxan-induced diabetic mice(group 3) with the reference

Page 17/36

drug(Glucophage) at a dose of 5mg/kg b.w. Groups 7, 8, and 9 were treated with ethyl acetate, methanol, andn-hexane at a particular dose of 100mg/kg b.w, and groups 4, 5, and 6 were treated with ethyl acetatefractions(EF-I, EF- II & EF-III) at a 100mg/kg b.w of P. guajava leaves respectively.

On the Fourteenth (14th) day of oral treatment of alloxan-induced diabetic mice with crude extracts andpartially puri�ed ethyl acetate fractions. The group 7 mice treated with ethyl acetate crude(100mg/kg b.w) leafextract of P. guajava had the highest percentage mean blood glucose with 43%(Mean±SEM), followed bymethanol, MC(35%), and n-hexane extract, HC (30%), EF I (20%) while EF II(18%) while EF III(4%) had leastpercentage blood glucose reduction. A statistical comparison of the anti-hyperglycemia effect of all treatmentgroups and positive controls (group 3) using a one way ANOVA (Dunnett's multiple) showed that each bar(Mean±SEM) was not signi�cantly different from the positive control group (59%) mean percentage bloodglucose reduction). This implies that each group treated with EF I, EF II, EF III, ethyl acetate, methanol, and n-hexane crude extract of P.guajava leaves had much signi�cant anti-hyperglyceamic effect compared with thestandard reference drug (Glucophage) administered at a dose of 5mg/kg b.w in the order of EC>MC>HC>EF-I>EF-II while the EF-III had least percentage blood glucose reduction.

DiscussionDiabetes mellitus is a metabolic condition categorized with several etiologies and chronic hyperglycemiacaused by disturbances of carbohydrate, fat, and protein metabolism resulting from defects in insulinsecretion, insulin action, or both(Wild et al., 2004). Diabetes mellitus is caused by the abnormality ofcarbohydrate metabolism, which is linked to low blood insulin levels or insensitivity of target organs to insulin(Lincy et al. 2016). It has been considered an incurable metabolic disorder affecting about 2.8% of the globalpopulation. Despite considerable progress in the treatment of diabetes by oral hypoglycemic agents, thesearch for newer drugs continues because the existing synthetic drugs have several limitations (Arumugam etal., 2013).

Alloxan treatment is a method widely used to induce hyperglycemia conditions (diabetes mellitus) in animalsusing alloxan, a glucose analog which is selectively toxic to pancreatic beta cells due to its accumulation inbeta cells. The mechanism of action of alloxan is enhanced by the presence of cysteine amino acid residuewhich contains two –SH groups that form a disul�de bond thereby inactivating enzyme. As a result of alloxanreduction, diuric acid is then re-oxidized back to alloxan, hence establishing a redox cycle for the generation ofreactive oxygen species and superoxide radicals (Wild et al., 2004). The cytotoxic action of alloxan ismediated mainly by these reactive oxygen species (ROS). Reactive oxygen species cause the fragmentation ofthe DNA of pancreatic islets, which occurs in beta cells exposed to alloxan monohydrate (Lincy et al., 2016).

The preliminary acute toxicity (LD50) study of ethyl acetate crude leaf extract of Psidium guajava was carriedout in different phases to establish the lethal oral dosage of ethyl acetate crude leaf extract of Psidiumguajava. The results showed that more than 500mg of ethyl acetate crude extract of the plant leaves was safefor consumption as a decoction.

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This study further evaluated the anti-hyperglycemic effects of methanol, ethyl acetate, and n-hexane crudeextracts and ethyl acetate bulked fractions of Psidium guajava Linn leaves in alloxan-induced diabetic miceand the results showed a signi�cant anti-hyperglyceamic effect in reduction of high blood glucose levels intest groups treated with a dose of 100mg/kg of each crude leaf extract of P. guajava as compared withnegative and positive controls.

However, the results of anti-hyperglycemic effect of ethyl acetate crude extract (EC) of P. guajava on alloxan-induced diabetic mice(group 7) exhibited much signi�cant[P<0.001, P<0.01; P<0.05]blood glucose reductionthroughout the oral treatment which lasted on the twelfth(12th) day of oral treatment, while alloxan-induceddiabetic mice(group 9) treated n-hexane crude extract (HC, 100mg/kg b.w) of Psidium guajava leaves showeda signi�cant[P<0.001, P<0.01; P<0.05]anti-hyperglyceamic control which lasted on 7th day of oral treatmentcompared with negative control(group 2). The alloxan-induced diabetic mice (group 8) treated with 100mg/kgof methanol extract of P. guajava leaves also showed signi�cant [P<0.001,P<0.01; P<0.05] anti-hyperglyceamiccontrol which lasted on the �fth(5th) day of oral administration compared with negative control(group 2).

The group 4 mice treated with ethyl acetate fraction I(EF-I) (100mg/kg b.w) showed a signi�cant [P<0.05] anti-hyperglycemia effect compared with the negative control(group 2) that lasted on the second (2nd) day of oraltreatment, while EF-II showed invariably a least signi�cant glucose control in the test group as compared withcontrol groups. Ef-iii exhibited a nonsigni�cant [P>0.05] hypoglycaemic effect as compared with the negativecontrols (group 2), despite a gradual decline in high blood glucose levels throughout the period of oraladministration. However, the pattern of response or decrease in random blood glucose concentration ofdiabetic treated groups differed signi�cantly over the period (days) of oral administration of each crude extractand ethyl acetate fractions respectively.

The high anti-hyperglycemic effect observed among different treatment groups with crude extracts and ethylacetate bulked fraction I(EF-I) administered at a speci�c dose of 100mg/kg b.w can be attributed to thepresence of rich antidiabetic bioactive compounds present in the crude extracts and bulked ethyl acetate fraction I (EF

I) of Psidium guajava leaves, since the qualitative phytochemical screening of crude leaf extracts, and ethylacetate bulked fraction I(EF I) showed a signi�cant presence of alkaloids, �avonoids, steroids, and terpenoidswhich have been identi�ed. In general, there is increasing biological knowledge on the speci�c modes of actionof medicinal plants in the treatment of diabetes, but most of the plants have been found to containsubstances like glycosides, alkaloids, terpenoids, and �avonoids that are frequently implicated as having anantidiabetic effect (Lincy et al., 2016). However, the methanol crude extract showed a moderate signi�cantdecrease in high blood glucose levels in treated mice (group 8), while ethyl acetate fractions II and III showed anonsigni�cant decrease in blood glucose level of alloxan-induced diabetic groups 5 and 6 respectively whencompared with diabetic untreated control (group 2) due to the little or trace amount of these bioactivecompounds. There are possible explanations for these �ndings, despite that there is no clear explanation ofthe in vitro mechanism of action of Psidium guajava crude extracts and ethyl acetate bulked fraction as anantidiabetic agent. It may be hypothesized that the major phytochemical constituent(s) of the crude extractand ethyl acetate bulked fractions may have delayed or reduced glucose absorption from the gastrointestinaltract into the circulatory system, through inhibition of carbohydrate digestion, or inhibition of Na+-glucose co-

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transporters and facilitated glucose transporters on the luminal (mucosal) side of the absorptive cells ofintestinal epithelial cells, or inhibition of Na+K+ - ATPase on the serosa side of intestinal epithelial cells.Moreover, the crude extracts of Psidium guajava leaves may have triggered the recovery of partially destroyedβ-cells or possibly Psidium guajava leaf crude extracts of n-hexane, ethyl acetate, and methanol and ethylacetate bulked fraction I, EF-I(1-75) may have initiated cell proliferation after induction of diabetes with alloxanmonohydrate (120mg/kg, i.p) in test mice. There was a nonsigni�cant reduction in blood glucose levels ofalloxan-induced diabetic mice (groups 5 and 6) treated with ethyl acetate bulk fractions, EF-II and EF-IIIcompared with negative and positive controls, despite the presence of trace amounts of these phytochemicalconstituents. The nonsigni�cant anti- hyperglyceamic effect showed by ethyl acetate bulk fractions, EF-II andEF-III were due to the loss of potent anti- bioactive compounds during the fractionation process and thesebioactive compounds have been implicated to elicit pharmacologic activity or synergistic that enhances anti-diabetic activity of fractions.

ConclusionThis study has shown that ethyl acetate crude leaf extract of Psidium guajava Linn exhibited a very highpotent antidiabetic effect followed by methanol, n-hexane crude leaf extract and ethyl acetate bulk fractionI(EF-I) by lowering the high blood glucose level in alloxan-induced diabetic treated mice compared withdiabetic untreated(negative control) in the order of EC>MC>HC>EF-I>EF-II>EF>III. The result �ndings showedthat the ethyl acetate bulked fractions, EF-II and EF-III exhibited a nonsigni�cant anti-hyperglycemia effectcompared with negative control mice (group 2), and despite the gradual decrease in high blood glucoseconcentration. In addition, a comparison of a percentage mean glucose reduction (Mean±SEM) of all diabetictreated groups with EF-I and all crude extracts administered at a dose of 100mg/kg b.w which showed a non-signi�cant difference from the positive control mice(group 3) treated with Glucophage (5mg/kg b.w), astandard anti-diabetic drug.

The phytochemical screening of methanol, ethyl acetate, n-hexane, crude leaf extract, and ethyl acetate bulkfractions of Psidium guajava showed a rich abundance of alkaloids, steroids, �avonoids, tannins, andsaponins and absence of glycosides and reducing sugar were observed in EF-I, EF-II, EF-III, EC, HC, and MC.The LD50 of ethyl acetate crude leaf extract of Psidium guajava was determined within the range of 1200-1500mg/kg / body weight, thus suggesting a wide safety margin for use in animal model experiments. Inconclusion, the resulting �ndings from this study have given credence to the use of Psidium guajava leaves asa traditional remedy for the treatment and management of diabetes mellitus.

Recommendation    In future studies, it is recommended that emphasis should be made towards the use of advancedchromatographic techniques for both fractionation and puri�cation of crude extracts of Psidium guajavaleaves.

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The use of advanced chromatographic techniques like High-Performance Liquid Chromatography (HPLC), GasChromatography Mass Spectrometry (GC-MS) and molecular spectroscopy should be used to further isolate,identify and characterize potent antidiabetic drug compound(s) in the puri�ed fraction that may be linked tothe anti-diabetic property of Psidium guajava. It is also necessary to carry out in vivo and in vitro antidiabeticstudies of anti-hyperglyceamic activity of ethyl acetate fractions of Psidium guajava leaves with varyingdoses (200mg/kg b.w, 400mg/kg b.w and 600mg/kg b.w) on a speci�c cultured cell line and alloxan-induceddiabetic mice over a prolonged duration, to assess the dose-dependent anti-hyperglycemic effect of eachfraction. The medicinal value of Psidium guajava plant should be given more attention towards developingnew antidiabetic drugs and other therapeutic drug researches.

DeclarationsAUTHORS’ CONTRIBUTIONS

This work was extensively conducted by Mr. Eze Uchenna Nwabunwanne under the supervision of AssociateProf. Anthonius A. Eze from the Department of Medical Biochemistry, Faculty of Basic Medical Sciences,University of Nigeria, Enugu, Nigeria. The Author conducted the literature search, experimental design and invivo bioassay, statistical analyses, proof-reading, and editing of the manuscript. All co-authors contributed tothe success of this research by sharing knowledge, research experience, and �nancial resources.

8.0 CONFLICTS OF INTEREST: We declare that there are neither con�icts of interests nor funding sponsors inexecuting this research paper.

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Figures

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Figure 1

Uninduced (Normal) control Induced but Untreated control Induced but treated control Bulk fraction 1 (1-75)

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Figure 2

Uninduced (Normal) control Induced but Untreated control Induced but treated control Bulk fraction II (76-150)

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Figure 3

Uninduced (Normal) control Induced but Untreated control Induced but treated control Bulk fraction III (151-250). Uninduced (Normal) control Induced but Untreated control Induced but treated control Ethyl acetatecrude extract

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Figure 4

Uninduced (Normal) control Induced but Untreated control Induced but treated control Methanol crude extract.Uninduced (Normal) control Induced but Untreated control Induced but treated control n-hexane crude extract

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Figure 5

Each Bar has a description

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Figure 6

Each Bar has a description